Table 1.
Oligonucleotide primers used for site-directed mutagenesis.
Figure 1.
The structure model of alkaline α-amylase from A. amylolytica and the structure of both template and modeled structure overlapping.
A: The model structure of alkaline α-amylase was constructed with the crystal structure of α-amylase AmyB (3bc9) as a template. The catalytic residues Asp248, Glu278, and Asp340 are shown in “CPK (Corey-Pauling-Koltum)” representation. The blue “sticks” indicated the mutant positions. The red “ball and stick” indicated the conserved regions. B: The overlapping of template and modeled α-amylase structure. The yellow is the template (AmyB) structure, and the other is the modeled structure of alkaline α-amylase.
Figure 2.
Oxidative stability of wild-type and mutant proteins.
The relative activity (%) was determined and compared with the activity without the addition of H2O2. A: Oxidative stability of the one-site mutant enzymes. B: Oxidative stability of eight five-site mutant enzymes.
Table 2.
Kinetic parameters of wild-type and one-point mutants.
Table 3.
Kinetic parameters of wild-type and eight five-point mutant enzymes.
Figure 3.
The circular dichroism (CD) and fluorescence spectroscopy of the wild-type and mutant proteins.
A: CD spectral; B: The fluorescence spectroscopy.
Figure 4.
The change of the salt bridge around active sites of mutant M145I-214A-229T-247T-317I.
The catalytic residues Asp248, Glu278 and Asp340 are shown in red “ball and stick” representation. The blue “sticks”: residues forming salt bridge around the active site. The α helices and β sheets are shown in red and cyan, respectively. A: The salt bridge around active sites of wild-type enzyme. The black “dotted line” is the salt bridge. B: The change of the salt bridge around active sites of the mutant. The newly formed salt-bridge between Arg 118 and Glu 104 is included in the “dotted frame”. The green “dotted line” is the new salt bridge formed after mutation.
Figure 5.
Effect of pH on activity and stability of eight five-site mutant enzymes.
A: Effect of pH on activity of mutants. B: Effect of pH on stability of mutants.
Figure 6.
Local hydrogen bonding network of the five-site mutants.
The active site is shown in a green CPK (Corey-Pauling-Koltun) representation. The yellow “ball and stick” indicate the conserved regions. The blue “stick” indicates the mutation site. The purple “stick” is the residue that forms new hydrogen bonding after mutation. The black “dotted line” is the hydrogen bonding. The α helices and β sheets are shown in red and cyan, respectively. A: M145A-214A-229A-247L-317I; B: M145I-214A-229T-247T-317I; C: M145A-214A-229T-247L-317I; D: M145A-214A-229T-247T-317I.
Figure 7.
Effect of temperature on the activity and stability of eight five-site mutant enzymes.
A: Effect of temperature on the activity of mutant enzymes. B: Effect of temperature on the stability of mutant enzymes. The inset presents the Arrhenius plot of the logarithm of the k values against the reciprocal of the absolute temperature (T). The values shown are activation energies calculated from the plot.
Figure 8.
Effects of surfactants and detergents on the eight five-site mutant enzymes.
A: Effects of surfactants on stability of mutants. B: Effects of detergents on activity of the mutants.